Restriction Endonuclease Fragments Of DNA Research Articles

Isolation and preliminary characterization of a new pathogenic iridovirus from redclaw crayfish Cherax quadricarinatus.

We report the preliminary characterization of a new iridovirus detected in diseased Cherax quadricarinatus collected from a farm in Fujian, China. Transmission electron microscopy identified numerous icosahedral particles (~150 nm in diameter) in the cytoplasm and budding from the plasma membrane of hematopoietic tissue cells. SDS-PAGE of virions semi-purified from the hemolymph of moribund C. quadricarinatus identified 24 proteins including a 50 kDa major capsid protein (MCP). By summing the sizes of DNA restriction endonuclease fragments, the viral genome was estimated to be ~150 kb in length. A 34 amino acid sequence deduced from a 103 bp MCP gene region amplified by PCR using degenerate primers targeted to MCP gene regions conserved among iridoviruses and chloriridoviruses was most similar (55% identity) to Sergestid iridovirus. Based on virion morphology, protein composition, DNA genome length, and MCP sequence relatedness, the virus identified has tentatively been named Cherax quadricarinatus iridovirus (CQIV). In addition, experimental infection of healthy C. quadricarinatus, Procambarus clarkii, and Litopenaeus vannamei with CQIV caused the same disease and high mortality, suggesting that CQIV poses a potential threat to cultured and wild crayfish and shrimp.

Molecular systematics ofBrassicaand allied genera in subtribes Brassicinae, Raphaninae, Moricandiinae, and Cakilinae (Brassicaceae, tribe Brassiceae); the organization and evolution of ribosomal gene families

DNA restriction endonuclease fragment analysis was used to obtain new information on the genomic organization of ribosomal DNA (rDNA) of Brassica and allied genera. The total genomic DNA of 95 accessions of 52 species representing 16 genera was restricted with six enzymes, and the restriction fragments were probed with three ribosomal clones (pTA71, Ver 18-6, and Ver 6-5). Eleven repeat unit length classes were recognized. The repeat unit size classes of 8.9 kb and 9.5 kb were observed most commonly, being represented in 17 and 14 species, respectively. The restriction enzyme SacI produced three to six (generally three) bands with detectable hybridization to the probe pTA71. This probe–enzyme combination indicated a remarkable uniformity amongst Brassica and allied genera in the coding region of repeat units. By contrast, an extensive size variation in the restriction fragments could be localized in the intergenic spacer (IGS) region. Eleven IGS-containing length variants were detected. Complex hybridization patterns, resulting from extensive repeat unit heterogeneity and taxon-specific methylation of one or more cleavage sites, were obtained with the EcoRI + pTA71 combination. The relative homologies between the coding regions were evident from the presence of 1.5 kb in all the taxa, and 0.4-, 1.3-, and 1.7-kb fragments in 33, 27, and 24 species, respectively. The SacI + pTA71 and EcoRI + pTA71 combinations were generally able to distinguish taxa both within and between genera. Three restriction endonuclease digests probed with three ribosomal clones yielded essentially identical fragmentation patterns across all the accessions within the cultivated species Brassica campestris, B. oleracea, and B. juncea. In B. napus, three and seven accessions exhibited restriction profiles similar to one and both diploid progenitor species, respectively. Overall, rDNA repeat unit length polymorphism showed good correlation with the cytodeme-based classification of Brassica and allied genera. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 157, 545–557.

Atypical Myxomatosis – Virus Isolation, Experimental Infection of Rabbits and Restriction Endonuclease Analysis of the Isolate

Atypical form of myxomatosis, which caused non-lethal and clinically mild disease in domestic rabbits 1 month after immunization with a commercially available vaccine MXT, is described. The isolated myxoma virus designated as Litovel 2 (Li-2) did not induce systemic disease following subcutaneous and intradermal applications in susceptible experimental rabbits but led to the immune response demonstrated by ELISA. No severe disease was induced in those Li-2 inoculated rabbits by challenge with the virulent strains Lausanne (Lu) or Sanar (SA), while the control animals showed nodular form of myxomatosis with lethal course of the illness. Restriction fragment length polymorphism (RFLP) of genomic DNA with KpnI and BamHI endonucleases was used for genetic characterization of the Li-2 isolate, the vaccine strain MXT and both virulent strains Lu and SA, respectively. In general, RFLP analysis has shown to be informative for inferring genetic relatedness between myxoma viruses. Based on restriction endonuclease DNA fragment size distribution, it was evident that the pathogenic strain SA is genetically related to the reference strain Lu and the isolate Li-2 is more related, but not identical, to the vaccination strain MXT.

Dependence of DNA steady-state electric birefringence on field strength

Abstract A major task in our work has been to calculate the dependence of DNA steady-state electric birefringence on field strength for a ‘hinged bent rod’ (HBR) composed of two rod-like arms of length L with a restoring force proportional to the angle of bending at the hinge. The system is considered to have a permanent dipole moment due to asymmetric charge distribution with respect to its centre of diffusion. The electric and optical polarizability tensors of HBR macroions are also calculated. To calculate the electric birefringence, the statistical averages of the differential optical polarizability per molecule in the directions parallel and perpendicular to the applied electric field are made over all conformations as well as over all spatial orientations and all the velocities in the presence of the electric field. Using this approach, numerical calculations are carried out to achieve a fitting of electric birefringence experimental curves versus the square of the electric field for the sonicated and fractioned DNA and restriction enzyme fragments of DNA. It is shown that the HBR macroion presents a reversal of the birefringence sign at low electric fields for certain chain lengths, electric polarizability and stiffness constant values, in agreement with the experimental results obtained for DNA. It is observed that the HBR model adequately represents DNA elastic properties.

Contamination of Marek's disease vaccine suspensions with Enterococcus faecalis and its possible role in amyloid arthropathy

Samples of hatchery air (hatcher and processing room), Marek's disease vaccine suspensions and injection needles collected during chick processing, revealed variable levels (< 500 to 106 colony forming units (cfu)/m3 air, < 10 to 106 cfu/ml vaccine suspension, and 9500 to 61000 cfu/needle) of Enterococcus faecalis contamination. This observation suggests a possible infection route in 1-day-old chickens through intramuscular vaccination of Marek's disease vaccine contaminated with arthropathic and amyloidogenic E. faecalis, which could lead to amyloid arthropathy. Pulsed-field gel electrophoresis (PFGE) DNA restriction endonuclease fragment analysis of E. faecalis strains obtained from two hatcheries revealed a predominant PFGE pattern in one hatchery, while one isolate with an almost identical PFGE pattern to an amyloid arthropathy inducing isolate was found.

Vitamin D receptor interactions with the rat parathyroid hormone gene: synergistic effects between two negative vitamin D response elements.

Vitamin D response elements (VDREs) that are required for negative regulation of rat parathyroid hormone (rPTH) gene expression have been characterized. Gel mobility shift assays using DNA restriction enzyme fragments and recombinant proteins for vitamin D and retinoic acid X receptors (VDR/RXR) revealed a sequence between -793 and -779 that bound a VDR/RXR heterodimer with high affinity (VDRE1). Furthermore, a lower affinity site (VDRE2) was detected that acted in combination with VDRE1 to bind a second VDR/RXR complex. As determined by ethylation interference analysis, the nucleotide sequence of VDRE1 consisted of GGTTCA GTG AGGTAC, which is remarkably similar to the sequence of the negative VDRE found in the chicken PTH (cPTH) gene. Using the same technique, VDRE2 was identified between positions -760 and -746 and contained the sequence AGGCTA GCC AGTTCA. Functional analysis was determined by transfection studies with plasmid constructs that expressed the gene for chloramphenicol acetyl transferase (CAT). The ability of the VDREs to regulate gene expression was tested in their native context with the rPTH promoter as well as when positioned immediately upstream from the cPTH promoter. With either plasmid construct, exposure to 10(-8)M 1,25(OH)2D3 resulted in a 60-70% decrease in CAT gene expression when both VDRE1 and VDRE2 were present. Examination of the individual VDREs showed that inhibition by 10(-8) M 1,25(OH)2D3 was only 35-40% when just VDRE1 was present. By itself, VDRE2 was even less effective, as significant inhibition of CAT activity (20%) was observed only in the presence of higher concentrations of 1, 25(OH)2D3 (10(-7)M) or when a plasmid vector that overexpressed the VDR protein was cotransfected. In conclusion, the rPTH gene contains two negative VDREs that act in concert to bind two RXR/VDR heterodimer complexes and that both VDREs are required for maximal inhibition by 1,25(OH)2D3.

Transformation and laccase mutant isolation inCoprinus congregatusby restriction enzyme-mediated integration

Coprinus congregatus did not show any growth on a minimal medium in the presence of phosphinothricin which inhibited glutamine synthetase. Genetic transformation to phosphinothricin resistance in C. congregatus was carried out successfully by restriction enzyme-mediated integration. The procedure was improved to yield 550 transformants per μg of DNA, and three laccase mutants were generated. The vector pBARGEM 7-1 which had the phosphinothricin resistance gene was detected in the restriction enzyme fragments of chromosomal DNA from the transformants by Southern hybridization. Transformants showed identical electrophoretic banding patterns but CL1430b had a faster moving band when analyzed by native PAGE.

Transformation and laccase mutant isolation in Coprinus congregatus by restriction enzyme-mediated integration

Coprinus congregatus did not show any growth on a minimal medium in the presence of phosphinothricin which inhibited glutamine synthetase. Genetic transformation to phosphinothricin resistance in C. congregatus was carried out successfully by restriction enzyme-mediated integration. The procedure was improved to yield 550 transformants per μg of DNA, and three laccase mutants were generated. The vector pBARGEM 7-1 which had the phosphinothricin resistance gene was detected in the restriction enzyme fragments of chromosomal DNA from the transformants by Southern hybridization. Transformants showed identical electrophoretic banding patterns but CL1430b had a faster moving band when analyzed by native PAGE.

Importance of the Intracytoplasmic Domain of the Simian Immunodeficiency Virus (SIV) Envelope Glycoprotein for Pathogenesis

SIVmac1A11 and SIVmac239 are nonpathogenic and pathogenic molecular clones in rhesus macaques, respectively. Although these viruses exhibit ∼98% nucleotide and amino acid sequence homology, differences are found in the length of the translation frames for several genes. SIVmac239 has a premature stop codon innef, whereas SIVmac1A11 has a premature stop codon invprand two premature stop codons in the intracytoplasmic domain of theenv-transmembrane (TM) subunit. Recombinant viruses, constructed through reciprocal exchange of large DNA restriction enzyme fragments between SIVmac1A11 and SIVmac239, were evaluated in adult rhesus macaques. Thisin vivoanalysis revealed that two or more regions of the SIVmac genome were essential for high virus load and disease progression (Marthaset al.,1993.J. Virol.67, 6047–6055). An important gap in knowledge remaining from this study was whether the premature stop codons inenv-TM of recombinant virus SIV1A11/239gag-env/1A11 (full-lengthvprandnef, two stop codons inenv-TM) reverted to coding tripletsin vivo. Here, we report that viral sequences in macaques, which succumbed to an AIDS-like disease after infection with SIV1A11/239gag-env/1A11, exhibited reversion of bothenv-TM stop codons. In addition, antibodies to the intracytoplasmic domain ofenv-TM were detected in macaques containing revertant virus and showing disease; this finding indicates that this domain of theenvglycoprotein was expressedin vivo. Thus selection for viral variants with full-lengthenv-TM demonstrated that the cytoplasmic domain of the SIVmacenvglycoprotein plays a role in viral persistence and immunodeficiency in primates.

Infrared MALDI mass spectrometry of large nucleic acids.

Mass spectrometry has become an increasingly important tool of high accuracy, efficiency, and speed for the routine analysis of nucleic acids. To make it useful for large-scale sequencing of genomic material as required for example in genotyping and clinical diagnosis, it is necessary to find approaches that allow the analysis of sequences much larger than the 100 nucleotides currently possible. Matrix-assisted laser desorption/ionization (MALDI) mass spectra of synthetic DNA, restriction enzyme fragments of plasmid DNA, and RNA transcripts up to a size of 2180 nucleotides are reported. The demonstrated mass accuracy of 1 percent or better and the sample requirement of a few femtomoles or less surpass all currently available techniques for the analysis of large nucleic acids. DNA and RNA can be analyzed with only a limited investment in sample purification.

Cloning of the genes for and characterization of the early stages of toluene and o-xylene catabolism in Pseudomonas stutzeri OX1.

In order to study the toluene and o-xylene catabolic genes of Pseudomonas stutzeri OX1, a genomic library was constructed. A 28-kb EcoRI restriction endonuclease DNA fragment, cloned into the vector plasmid pLAFR1 and designated pFB3401, permitted Pseudomonas putida PaW340 to convert toluene and o-xylene into the corresponding meta-ring fission products. Physical and functional endonuclease restriction maps have been derived from the cloned DNA fragment. Further subcloning into and deletion analysis in the Escherichia coli vector pGEM-3Z allowed the genes for the conversion of toluene or o-xylene into the corresponding catechols to be mapped within a 6-kb region of the pFB3401 insert and their direction of transcription to be determined. Following exposure to toluene, E. coli cells carrying this 6-kb region produce a mixture of o-cresol, m-cresol, and p-cresol, which are further converted to 3-methylcatechol and 4-methylcatechol. Similarly, a mixture of 2,3-dimethylphenol and 3,4-dimethylphenol, further converted into dimethylcatechols, was detected after exposure to o-xylene. The enzyme involved in the first step of toluene and o-xylene degradation exhibited a broad substrate specificity, being able to oxidize also benzene, ethylbenzene, m-xylene, p-xylene, styrene, and naphthalene. Deletions of the 6-kb region which affect the ability to convert toluene or o-xylene into the corresponding methylphenols compromise also their further oxidation to methylcatechols. This suggests that a single enzyme system could be involved in both steps of the early stages of toluene and o-xylene catabolism.

Relationships between populations of Pseudomonas syringae pv. persicae determined by restriction fragment analysis

Pseudomonas syringae pv. persicae, the pathogen causing bacterial decline of stone‐fruit, was first noted almost simultaneously in France and in New Zealand, together with a closely related pathogen from myrobalan plum in England. The relative similarity of 31 strains from these three countries was examined by comparing DNA restriction endonuclease fragment patterns. Fragment patterns produced from digested DNA samples which were electrophoresed on polyacrylamide gels and visualized by silver‐staining, were analysed using the software package Gelcompar. The fragment patterns produced by strains from France and England formed homogeneous but separate groups, while those from New Zealand were relatively heterogeneous. This finding suggests that the New Zealand population of P.s. persicae is older than those found in Europe. Problems in explaining the distribution of the pathogen are discussed.

End-labeling of DNA fragments.

AbstractEnd-labeling is a rapid and sensitive method for radioactively, or nonisotopically, labeling DNA fragments and is useful where there is too little DNA to visualize by ethidium bromide staining. This can be a serious problem when separating small fragments by polyacrylamide gel electrophoresis (see Chapter 13). End-labeling has the second advantage that it can be used to produce fragments labeled at one end. Because all of the enzymes employed are specific to either the 5′ or 3′ termini of DNA molecules, it will only incorporate the label once per DNA strand. When double-stranded DNA restriction enzyme fragments are used, the label is incorporated at both ends, but can be removed from one by a second restriction enzyme digestion. This works well with DNA fragments cloned into polylinkers because one end can be removed as a tiny piece of DNA, making subsequent purification easier. These single labeled molecules can help to order restriction enzyme fragments and are a prerequisite for Maxam-Gilbert DNA sequencing (see Chapter 53). End-labeled synthetic oligonucleotides have numerous applications, such as short DNA sequence-specific probes for mutation screening (1); probes for gel retardation and Southwestern assays (2); and for sequencing PCR products where the samples are contaminated with other nucleotides (see Chapter 49).KeywordsExonuclease ActivityKinase ReactionCalf Intestinal Alkaline PhosphataseRestriction Enzyme FragmentLabel NucleotideThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Nucleotide sequence of the gene encoding novel delta-endotoxin from Bacillus thuringiensis serovar japonensis strain Buibui specific to scarabaeid beetles

A new isolate of Bacillus thuringiensis serovar japonensis strain Buibui, which was specific to scarab beetles (M. Ohba et al., Lett. Appl. Microbiol. 14:54, 1992), was shown to have a 130-kDa insecticidal crystal protein (ICP) (H. Hori et al., J. Appl. Bacteriol. 76:307, 1994). ClaI restriction enzyme fragments of total cell DNA of the isolate were cloned into E. coli (Sato et al., Curr. Microbiol. 28:15, 1994). Whole 3480-bp nucleotide sequence of the gene encoding 130-kDa ICP was determined, and the molecular weight of the ICP was estimated to be 130,424. The strongly conserved five blocks that occur in almost all ICP genes of B. thuringiensis were detected in the ORF with the same order and almost the same intervals as elsewhere. The amino acid sequence homologies of the whole ICP or N-terminus half portion to that of the CryIIIA, B, C, D, and CryV were about 35%.

Ribosomal DNA repeat unit polymorphism in 49 Vicia species

DNA restriction endonuclease fragment analysis was used to obtain new information on the genomic organization of Vicia ribosomal DNA (rDNA), more particularly among V. faba and its close relatives and the taxa within three (Narbonensis, Villosa, Sativa) species' complexes. Total genomic DNA of 90 accessions representing 49 Vicia species was restricted with 11 enzymes, and the restriction fragments were probed with three ribosomal clones. Twenty-eight repeat unit length classes were identified. The number of length classes (1-2) per accession did not correspond to the number of nucleolar organizing regions (NORs). The number of rRNA genes was independent of the 2C nuclear DNA amount present in the taxon. Each of the 90 accessions had 2 (rarely 1)-4 DraI sites. Those taxa with the same number of DraI sites generally could be distinguished from each other by different configurations. Probing of the DNA samples digested with tetranucleotide recognition restriction endonucleases emphasized differences between divergent spacer regions and enabled relative homologies between the coding regions to be established. Overall, rDNA restriction site variation among the species showed a good correlation with taxonomic classification. The rDNA analysis indicated evolutionary relatedness of the various taxa within the Narbonensis species complex. rDNA diversity within two other species complexes (Villosa, Sativa), on the other hand, was more extensive than expected. With few exceptions, data on the two complexes give evidence of taxon-specific divergences not seen with other approaches. The restriction site variability and repeat length heterogeneity in the rDNA repeat exhibited startling differences between V.faba and its close wild relatives included in the Narbonensis species complex. This analysis provides new evidence that none of the species within the complex can be considered to be putative allies of broad bean.

Characterization of intergenic spacers in two rrn operons of Enterococcus hirae ATCC 9790

Two DNA restriction enzyme fragments coding for the 3' termini of 16S rRNA, the 5' termini of 23S rRNA, and the intergenic spaces between them in Enterococcus hirae ATCC 9790 were cloned and sequenced. The intergenic space of one of these genes contains a tRNA(Ala) sequence, whereas the other does not. Nevertheless, the intergenic spaces contain several regions that exhibit high levels of sequence homology and are capable of forming structures with similar base pairs. An analysis of Southern blots of chromosomal DNA cut with one and two restriction enzymes indicated that E. hirae has a total of six rrn operons.

Isolation and characterization of encephalitic bovine herpesvirus type 1 isolates from cattle in North America

Summary Nine cns bovine herpesvirus type 1 (bhv-1) isolates, recovered from bovine brain samples submitted to the Texas Veterinary Medical Diagnostic Laboratories from 1974-1989, were compared by analyzing their dna restriction endonuclease (re) fragment migration pattern. Seven had pattern similar to that of the respiratory bhv-1 Cooper strain. The remaining 2 isolates, however, had variant patterns, similar to that of each other, but completely different from patterns for the other 7. The re patterns of these 2 variants were similar to published re patterns for 2 encephalitic or neuropathogenic bhv-1 strains — the Australian N-569 strain and the Argentine A-663 strain. One of the Texas encephalitic variants (No. 30326) was isolated from the cns of a calf that died during an epizootic of encephalitis in 1974. The other, designated TX-89, was isolated in 1989 from the cns of a 7-month-old feedlot steer with acute fatal encephalitis. Microscopic lesions of encephalitis with neuronal degeneration and intranuclear inclusions were observed for 3 of the 9 isolates, the 2 variant isolates (No. 30326 and TX-89), and a respiratory isolate. The remaining 6 cns isolates, all respiratory subtypes, were recovered from cattle that did not have clinical cns disease or gross or microscopic cns lesions; in 5 of these cattle, virus was recovered from at least 1 other organ (lungs) besides the cns. We conclude that the cns of calves can be naturally infected with 2 distinct bhv-1 subtypes, the respiratory and the encephalitic, and that the encephalitic subtype (subtype 3 or bhv-1.3) has been present in Texas cattle since at least 1974.

The genomes of simian varicella virus and varicella zoster virus are colinear

Simian varicella virus (SVV) causes an exanthematous disease in non-human primates which is clinically similar to varicella zoster virus (VZV) infection of humans. In this study, the genetic relatedness of SVV and VZV was confirmed and the location of SVV DNA sequences homologous to VZV restriction endonuclease (RE) fragments and viral genes was determined. VZV DNA RE fragments representing 98.3% of the VZV genome were 32P-labeled and hybridized to RE digested, immobilized SVV DNA. Homologous sequences were located throughout the viral DNAs in similar map positions, indicating a colinear relationship between the VZV and SVV genomes. 32P-labeled VZV glycoprotein (gp I, II, III, and IV) and gene 62 DNA probes also hybridized to SVV DNA in a colinear manner. The results suggest that the location of specific SVV genes may be predicted from the known map positions of homologous VZV genes. This study provides further support for SVV infection of non-human primates as a model for VZV infection of humans.

The replication origin of proplastid DNA in cultured cells of tobacco

When tobacco suspension culture line BY2 cells in stationary phase are transferred into fresh medium, replication of proplastid DNA proceeds for 24 h in the absence of nuclear DNA replication. Replicative intermediates of the proplastid DNA concentrated by benzoylated, naphthoylated DEAE cellulose chromatography, were radioactively labelled and hybridized to several sets of restriction endonuclease fragments of tobacco chloroplast DNA. The intermediates hybridized preferentially to restriction fragments in the two large inverted repeats. Mapping of D-loops and of restriction fragment lengths by electron microscopy permitted the localization of the replication origin, which was close to the 23S rRNA gene in the inverted repeats. The replication origins in both segments of the inverted repeat in tobacco proplastid DNA were active in vivo.

Nosocomial Outbreak of Respiratory Tract Colonization withMycobacterium fortuitum: Demonstration of the Usefulness of Pulsed-Field Gel Electrophoresis in an Epidemiologic Investigation

Between August 1989 and January 1990, 16 patients on an alcoholism rehabilitation ward (ARW) developed positive sputum cultures for Mycobacterium fortuitum. During a 2-wk surveillance period, six of 43 ARW patients but none of 20 staff members had positive sputum cultures. In addition, none of 54 patients and staff on an adjacent ward sharing the same ice machine and water supply had positive cultures, and none of 92 acid-fast bacilli cultures performed on all sputum specimens from all other inpatient sources during the same 2-wk period were positive. The only exposure factor common to all cases was the use of one or both of the ward showers. Compared with 36 ARW control patients, cases were more likely to report clinical criteria for chronic bronchitis (odds ratio, 6.6; 95% confidence interval, 1.5 to 28.6; p = 0.02). Using phenotype analysis, plasmid profiles, and pulsed-field gel electrophoresis of large genomic DNA restriction enzyme fragments, the 16 case isolates were found to be identical. This strain of M. fortuitum was also cultured from a tap connected to the water line supplying the ARW showers, but not from the showers themselves. No further cases were identified after the showers were disconnected and decontaminated. To our knowledge, this is the first clinical use of pulsed-field gel electrophoresis for genetic comparison of mycobacterial strains. It demonstrates the important potential of this technique for studying the epidemiology of mycobacterial infections. Showers should be considered a possible source of nosocomial respiratory tract colonization with M. fortuitum.